Caprine beta-mannosidosis is a well-characterized glycoprotein catabolic disorder associated with the deficiency of beta-mannosidase activity, the lysosomal storage of uncatabolized oligosaccharide substrates, and severe neurological dysfunction. Beta-Mannosidase characterization, gene cloning and expression will be performed to: (1) better define lysosomal beta-mannosidase processing, transport and regulation; and (2) delineate the molecular defect and pathogenetic mechanisms in beta-mannosidosis. SPECIFIC AIM 1. CHARACTERIZATION OF THE BETA-MANNOSIDASE GENE PRODUCT. Large scale purification of beta-mannosidase has made it possible to obtain partial amino acid sequence. the amino acid sequence determined from these studies will be used to clone beta-mannosidase. synthesis, processing, timing of glycosylation and transport of beta-mannosidase peptides will be studied by metabolic labeling of cultured cells. To supplement these processing studies, N-terminal amino acid sequence analysis of beta-mannosidase peptides will be performed. Kinetic parameters, using the presumed natural substrates and purified enzyme, will be performed. SPECIFIC AIM 2. CLONING AND IN VITRO EXPRESSION OF BETA-MANNOSIDASE. cDNA for beta-mannosidase will be cloned from a bovine cDNA library using oligonucleotide screening. A full length cDNA clone will be constructed and used to express active beta-mannosidase. The molecular defect will be determined by assessing alterations in the DNA and mRNA from affected animals. These studies will provide important new information about lysosomal beta-mannosidase as well as the gene defect in beta-mannosidosis animals. This information will contribute to the long range goals of gene replacement therapy in an animal model of early- onset neurodegenerative lysosomal storage disease. New insights into the pathogenesis of beta-mannosidosis at clinical, physiological, cellular, metabolic and molecular levels are expected outcomes of this investigation.